Choice of a Column for Gas Chromatography-Mass Spectrometry of Fatty Acid Derivatives
The choice of columns for GC analysis in general is discussed in a separate web document or in the appropriate chapter in our online book 'Gas Chromatography and Lipids'. As a rule, there are few problems in using most of the commercial columns and stationary phases in widespread use for the analysis of methyl ester derivatives of fatty acids in the range C14 to C22 by mass spectrometry. However, problems can arise with samples that are a little out of the ordinary and especially when 3-pyridylcarbinol (picolinyl) esters, pyrrolidides and 4,4-dimethyloxazoline (DMOX) derivatives must be analysed by GC-MS. Bleeding of the stationary phase can then lead to troublesome backgrounds.
A number of manufacturers now make columns with cross-linked highly stable phases specifically for use with mass spectrometry. We have made extensive use of a column of Supelcowax 10™ (25 m in length; from Supelco-Sigma Inc.) in our laboratory. It is typical of columns of the Carbowax type in its elution properties, and can cover with ease a wide range of fatty acid methyl esters or DMOX derivatives (up to C30 in chain length).
The figure above illustrates a typical chromatogram of the fatty acids of human erythrocytes as methyl esters on a column of the Carbowax type (25 m) with a flame-ionization detector. Excellent separations are achieved of fatty acid esters of a given chain length that differ both by degree of unsaturation and in the positions of the double bonds. For example, two isomers of 18:1 and of 18:3 are separated, as are three isomers of 20:3, two of 20:4 and two of 22:5. With the methyl esters of the more common families of polyunsaturated fatty acids, the shorter the distance between the last double bond and the end of the molecule, the longer the retention time of the isomer. Even better separations of positional isomers might be obtained with a more polar phase, though the order of elution relative to components of a different chain length might vary and there can be other problems of components overlapping. For example, we have used a 100 m column of CPSil-88TM for methyl esters of C14 to C22 fatty acids without difficulty. By a judicious use of two or more columns differing in polarity in this way, most of the fatty acids of metabolic importance can be separated and estimated.
3-Pyridylcarbinol esters and pyrrolidides are more polar or have higher molecular weights, and the Supelcowax column can cope with the common range up to about C22. When a more polar phase is required, we have used a column of BPX-70™ (from SGE Ltd). Of course, other manufacturers will also have columns available that fully meet the requirements for GC-MS, but we no experience of any other than those listed.
Finally, we have a requirement for a column coated with a low polarity silicone phase, because of its high thermal stability. Such columns lack the resolution of phases of higher polarity, but are adequate for many purposes. We use a DB5™ column for fatty acids of longer than usual chain length (especially as picolinyl esters), for hydroxy fatty acids, and for other lipids of high molecular weight, including sterols and waxes. It is essential for any fatty acid derivative or other lipid that has been silylated.
It is not always possible to expect the same quality of resolution in mass spectrometry applications as with a flame-ionization detector (FID), mainly because of the greater dead volume in a mass spectrometer in comparison with an FID. This depends largely on the specific instrument in use, so further comment is impossible here. Also, it is not possible to expect the same resolution with 3-pyridylcarbinol esters and pyrrolidides as with methyl esters and DMOX derivatives, because of the high molecular weight and polarity of the former. However, pyrrolidides do have some interesting GC properties in their own right (see the appropriate web page).
- Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Woodhead Publishing and now Elsevier) (2010) - see Science Direct.
Updated: May 5th, 2014